iFlpMosaics enable the multispectral barcoding and high-throughput comparative analysis of mutant and wild-type cells

Area of Science:

• Genetics
• Developmental Biology
• Molecular Biology

Background:

• Understanding gene function requires comparing mutant and control cells.
• Current methods for creating genetic mosaics in mice have limitations in accuracy, throughput, and barcoding diversity.
• Comparing cells from different animals introduces confounding epigenetic and microenvironmental variations.

Purpose of the Study:

• To develop a novel toolkit for inducing and analyzing genetic mosaics in mice.
• To enable accurate determination of gene cell-autonomous function and modeling of somatic mutation-driven diseases.
• To overcome limitations of existing technologies for genetic mosaic induction.

Main Methods:

• Introduction of the iFlpMosaics toolkit, featuring new genetic tools and mouse lines.
• Utilizing recombinase-dependent ratiometric induction for controlled gene manipulation.
• Employing single-cell clonal tracking of multiple fluorescently labeled cells.
• Profiling labeled cells via multispectral imaging, flow cytometry, and single-cell RNA sequencing.

Main Results:

• The iFlpMosaics toolkit facilitates the induction of genetic mosaics with high accuracy and throughput.
• Enables single-cell clonal tracking within the same tissue microenvironment and time window.
• Allows for the analysis of any quiescent or progenitor cell, and any combination of floxed genes.
• Provides a method for precise profiling of labeled cells using advanced imaging and sequencing techniques.

Conclusions:

• iFlpMosaics significantly advances the ability to study gene function and disease mechanisms.
• The toolkit enables a more accurate understanding of how genetic mutations impact single-cell biology.
• Facilitates research in tissue development, homeostasis, and disease modeling through precise genetic mosaic analysis.